Methods for recovering functions of a damaged tissue caused by a variety of reasons are roughly classified as transplant therapy and regeneration therapy. In transplant therapy, organs provided from a donor are transplanted to replace a recipient's organs to restore lost functions of a living body.
In contrast, in regeneration therapy cells or tissues from several persons including the patient are cultured to be processed to form the organs. These cells replace those with injuries to repair or regenerate them by using the stem cells.
At present, three types of cells such as a human somatic stem cell, a human embryonic stem cell (ES cell), and human induced pluripotent stem cell (iPS cell) are applied or said that they are applicable to the regeneration therapy.
Here, the human somatic stem cell has been already used in a study. It exists in adult tissues and can only differentiate into specified tissue, or organs. Note that “mesenchymal cell” existing in a bone marrow or adipose cells can unusually be differentiated into a variety of tissues such as a bone, a cartilage, an artery and vein. When such somatic cells are used, use of autologous cell prevents immune rejection, and gives good engraftment. Furthermore, there is no report that long term cultivation of these stem cell results in transforming them into tumor cells.
On the other hand, it is known that cells to be differentiated are limited to some extent, the cell collection from the human tissue accompanies invasion, the tissue types to be formed by the differentiated cells are limited in some extent, and capable passage number are limited to forty and several times, namely, 100 to 200 days being calculated in terms of day numbers.
A human embryonic stem cell (ES cell) is the stem cell which is derived from the “inner cell mass” in surplus embryo (blastocyst) obtained from regeneration therapy and the like to be cultured. Since it forms teratoma as an index for its pluripotency, it may be differentiated into any one of the tridermic phases. There is a report that it could be differentiated into myocardium, nerve, and retina. Since the embryonic stem cell (ES) is an immortalized cell strain, one strain among them is continuously cultured endlessly. Then, under the proper culture conditions, a product may have stable properties as the cell is manufactured on a large scale.
On the other hand, it utilizes a fertilized ovum so that it requires strict handling not so as to cause ethical problems. Also, they are basically hetero-transplantation; it requires means to prevent a rejection response by an immune response. Further, it is known that they requires heterologous cell or serum, when the cells ae cultured; and they easily form the teratoma (benign tumor), if the very few number of undifferentiated cells are mixed in a transplanted regenerated tissue.
Human induced pluripotent stem (iPS) cell is established by introducing a part of genes, which are specifically expressed in the ES cell into a human adult cell (dermis and the like). If autologous iPS cells are used, the immune rejection problem does not occur; the differentiation technique is employed for ES cell as is. Finally, the induced pluripotent stem (iPS) cell does not use the fertilized ovum which is used in ES cells, but uses an adult tissue to enable to produce a cell having the same quality as that of the embryonic stem cell. There is no problem of the rejection response by the immune response when the autologous iPS cell is utilized.
On the other hand, it is known that the iPS cells are easily turned into benign tumor cells or cancer cells (embryo cell tumor) and the ratio of the cells established as iPS cells is low; because the morphologically similar cells to ES cells are selected from the whole cells to which genes are introduced.
The use of the stem cell itself in the regeneration therapy has the problems described above. Therefore, a method for using biological factors produced by the various stem cells, not but using the stem cells by themselves, for example, a variety of growth factors are disclosed (WO 2011/118795, herein below, it is referred to as a “prior art 1”). In particular, the prior art 1 (WO 2011/118795) discloses a composition for treating the damaged area comprising a culture sup of the stem cell derived from such as human exfoliated dens deciduous and the like, namely the culture sup including the growth factors such as vein epithelial cell growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transformation growth factor-β (TGF-β) and the like.
However, since the stem cell derived from the dental pulp used is not an established cell line, the culture sup of the stem cell of the interest cannot be obtained, unless the stem cell is prepared at a time of use, or a cryopreserved sample is melted to grow the cells. Therefore, there is the problem that it takes time to obtain the culture sup.
In general, among the cultured cells, the cell strain established from a normal cell cannot divide after 50 to 60 times of passage, and the cell dies. Naturally, ratios in the composition of the biological factors produced by the cultivated cells are changing in a time-dependent manner. Therefore, there is the difficult problem to obtain the culture supernatant having stable ratios of them, if the lined cells being capable of unlimited growth.
On the other hand, a typical cell being capable of unlimited growth is a cancer cell. This is caused by the cancer cell which is out of control to grow unlimitedly, although such growth and division of the normal cell is under control of the living body. Therefore, even if the cell may grow unlimitedly, such cancer cells will not be of any use, because the cells may produce biological factors harmful for the living body.
As described above, there is a strong need to establish the immortalized cell which is not cancerous, but grow unlimitedly.
In order to use the supernatant as the pharmaceutical preparation, the immortal stem cell would be able to continuously produce the certain biological factors.